Jan Wijnholds

The multidrug resistance protein family.

The human multidrug resistance protein (MRP) family contains at least six members: MRP1, the godfather of the family and well known as the multidrug resistance protein, and five homologs, called MRP2-6. In this review, we summarize what is known about the protein structure, the expression in tissues, the routing in cells, the physiological functions, the substrate specificity, and the role in multidrug resistance of the individual members of the MRP family.

The human multidrug resistance protein MRP4 functions as a prostaglandin efflux transporter and is inhibited by nonsteroidal antiinflammatory drugs

Prostaglandins are involved in a wide variety of physiological and pathophysiological processes, but the mechanism of prostaglandin release from cells is not completely understood. Although poorly membrane permeable, prostaglandins are believed to exit cells by passive diffusion. We have investigated the interaction between prostaglandins and members of the ATP-binding cassette (ABC) transporter ABCC [multidrug resistance protein (MRP)] family of membrane export pumps. In inside-out membrane vesicles derived from insect cells or HEK293 cells, MRP4 catalyzed the time- and ATP-dependent uptake of prostaglandin E1 (PGE1) and PGE2. In contrast, MRP1, MRP2, MRP3, and MRP5 did not transport PGE1 or PGE2. The MRP4-mediated transport of PGE1 and PGE2 displayed saturation kinetics, with Km values of 2.1 and 3.4 μM, respectively. Further studies showed that PGF1α, PGF2α, PGA1, and thromboxane B2 were high-affinity inhibitors (and therefore presumably substrates) of MRP4. Furthermore, several nonsteroidal antiinflammatory drugs were potent inhibitors of MRP4 at concentrations that did not inhibit MRP1. In cells expressing the prostaglandin transporter PGT, the steady-state accumulation of PGE1 and PGE2 was reduced proportional to MRP4 expression. Inhibition of MRP4 by an MRP4-specific RNA interference construct or by indomethacin reversed this accumulation deficit. Together, these data suggest that MRP4 can release prostaglandins from cells, and that, in addition to inhibiting prostaglandin synthesis, some nonsteroidal antiinflammatory drugs might also act by inhibiting this release.

Multidrug resistance protein 1 protects the choroid plexus epithelium and contributes to the blood-cerebrospinal fluid barrier

Multidrug resistance protein 1 (MRP1) is a transporter protein that helps to protect normal cells and tumor cells against the influx of certain xenobiotics. We previously showed that Mrp1 protects against cytotoxic drugs at the testis-blood barrier, the oral epithelium, and the kidney urinary collecting duct tubules. Here, we generated Mrp1/Mdr1a/Mdr1b triple-knockout (TKO) mice, and used them together with Mdr1a/Mdr1b double-knockout (DKO) mice to study the contribution of Mrp1 to the tissue distribution and pharmacokinetics of etoposide. We observed increased toxicity in the TKO mice, which accumulated etoposide in brown adipose tissue, colon, salivary gland, heart, and the female urogenital system. Immunohistochemical staining revealed the presence of Mrp1 in the oviduct, uterus, salivary gland, and choroid plexus (CP) epithelium. To explore the transport function of Mrp1 in the CP epithelium, we used TKO and DKO mice cannulated for cerebrospinal fluid (CSF). We show here that the lack of Mrp1 protein causes etoposide levels to increase about 10-fold in the CSF after intravenous administration of the drug. Our results indicate that Mrp1 helps to limit tissue distribution of certain drugs and contributes to the blood-CSF drug-permeability barrier.

Characterization of the MRP4- and MRP5-mediated Transport of Cyclic Nucleotides from Intact Cells

Cyclic nucleotides are known to be effluxed from cultured cells or isolated tissues. Two recently described members of the multidrug resistance protein family, MRP4 and MRP5, might be involved in this process, because they transport the 3′,5′-cyclic nucleotides, cAMP and cGMP, into inside-out membrane vesicles. We have investigated cGMP and cAMP efflux from intact HEK293 cells overexpressing MRP4 or MRP5. The intracellular production of cGMP and cAMP was stimulated with the nitric oxide releasing compound sodium nitroprusside and the adenylate cyclase stimulator forskolin, respectively. MRP4- and MRP5-overexpressing cells effluxed more cGMP and cAMP than parental cells in an ATP-dependent manner. In contrast to a previous report we found no glutathione requirement for cyclic nucleotide transport. Transport increased proportionally with intracellular cyclic nucleotide concentrations over a calculated range of 20–600 μm, indicating low affinity transport. In addition to several classic inhibitors of organic anion transport, prostaglandins A1 and E1, the steroid progesterone and the anti-cancer drug estramustine all inhibited cyclic nucleotide efflux. The efflux mediated by MRP4 and MRP5 did not lead to a proportional decrease in the intracellular cGMP or cAMP levels but reduced cGMP by maximally 2-fold over the first hour. This was also the case when phosphodiesterase-mediated cyclic nucleotide hydrolysis was inhibited by 3-isobutyl-1-methylxanthine, conditions in which efflux was maximal. These data indicate that MRP4 and MRP5 are low affinity cyclic nucleotide transporters that may at best function as overflow pumps, decreasing steep increases in cGMP levels under conditions where cGMP synthesis is strongly induced and phosphodiesterase activity is limiting.

Noninvasive, In Vivo Assessment of Mouse Retinal Structure Using Optical Coherence Tomography

Background Optical coherence tomography (OCT) is a novel method of retinal in vivo imaging. In this study, we assessed the potential of OCT to yield histology-analogue sections in mouse models of retinal degeneration. Methodology/Principal Findings We achieved to adapt a commercial 3rd generation OCT system to obtain and quantify high-resolution morphological sections of the mouse retina which so far required in vitro histology. OCT and histology were compared in models with developmental defects, light damage, and inherited retinal degenerations. In conditional knockout mice deficient in retinal retinoblastoma protein Rb, the gradient of Cre expression from center to periphery, leading to a gradual reduction of retinal thickness, was clearly visible and well topographically quantifiable. In Nrl knockout mice, the layer involvement in the formation of rosette-like structures was similarly clear as in histology. OCT examination of focal light damage, well demarcated by the autofluorescence pattern, revealed a practically complete loss of photoreceptors with preservation of inner retinal layers, but also more subtle changes like edema formation. In Crb1 knockout mice (a model for Lebers congenital amaurosis), retinal vessels slipping through the outer nuclear layer towards the retinal pigment epithelium (RPE) due to the lack of adhesion in the subapical region of the photoreceptor inner segments could be well identified. Conclusions/Significance We found that with the OCT we were able to detect and analyze a wide range of mouse retinal pathology, and the results compared well to histological sections. In addition, the technique allows to follow individual animals over time, thereby reducing the numbers of study animals needed, and to assess dynamic processes like edema formation. The results clearly indicate that OCT has the potential to revolutionize the future design of respective short- and long-term studies, as well as the preclinical assessment of therapeutic strategies.

MRP6 (ABCC6) detection in normal human tissues and tumors

M RP6 (ABCC6) is a member of the subfamily of the multidrug resistance proteins (MRPs, reviewed by Borst et al, 2000), but its putative role in multidrug resistance (MDR, reviewed by Moscow et al, 1997) is still under investigation. Closely related proteins such as MDR1 P-glycoprotein (P-gp, ABCB1, reviewed by Ambudkar et al, 1999), breast cancer resistance protein (BCRP, ABCG2; Doyle et al, 1998), and MRP1, -2, and -3 (ABCC1–3) are established MDR transporters. The exact range of substrates for MRP6 has not yet been determined, but a preliminary report suggested that MRP6 may be involved in the transport of certain anticancer agents, including anthracyclines and epipodophyllotoxins (M.G. Belinsky et al, Proceedings of the AACR, abstract 1510, 2001). Recently it was found that mutations in the MRP6 gene cause pseudoxanthoma elasticum (PXE), an inheritable disorder of the connective tissue involving impaired visual acuity, skin lesions, and cardiovascular complications (Bergen et al, 2000). The expression of MRP6 in normal human tissues has only been studied at the mRNA level. High MRP6 mRNA levels were reported in liver and kidney, whereas low expression was found in a range of other tissues, including lung, intestines, retina, skin, and vessel walls (Bergen et al, 2000; Kool et al, 1999). To study MRP6 at the protein level, three rat Mabs (M6II-7, M6II-21, and M6II-31) were generated from rats immunized with a fusion protein containing amino acids 764 to 964 of human MRP6 (FP M6II), according to described methods (Scheffer et al, 2000). Reactivity of these Mabs to full length MRP6 protein was shown in Western blots with fractions of MRP6-overexpressing HEK 293 cells. All Mabs reacted with the approximately Mr 180,000 MRP6 protein, that migrated slightly faster than the related MRP2 protein (ABCC2), as detected with the M2III-6 Mab (Scheffer et al, 2000) in a control cell line (Fig. 1). Isotype specific secondary antibodies (Nordic, Tilburg, The Netherlands) showed that M6II-7 and M6II-31 were both of IgG2a subclass, whereas Mab M6II-21 was of IgG1 subclass. Lack of cross reactivity of M6II-7, M6II-21, and M6II-31 with MDR1 P-gp or MRP1, -2, -3, -4, and -5 family members, was concluded from staining results from cytospin preparations of several cell lines and Western blot experiments with protein fractions

Crumbs homologue 1 is required for maintenance of photoreceptor cell polarization and adhesion during light exposure

Loss of Crumbs homologue 1 (CRB1) function causes either the eye disease Leber congenital amaurosis or progressive retinitis pigmentosa, depending on the amount of residual CRB1 activity and the genetic background. Crb1 localizes specifically to the sub-apical region adjacent to the adherens junction complex at the outer limiting membrane in the retina. We show that it is associated here with multiple PDZ protein 1 (Mupp1), protein associated with Lin-7 (Pals1 or Mpp5) and Mpp4. We have produced Crb1-/- mice completely lacking any functional Crb1. Although the retinas are initially normal, by 3-9 months the Crb1-/- retinas develop localized lesions where the integrity of the outer limiting membrane is lost and giant half rosettes are formed. After delamination of the photoreceptor layer, neuronal cell death occurs in the inner and outer nuclear layers of the retina. On moderate exposure to light for 3 days at 3 months of age, the number of severe focal retinal lesions significantly increases in the Crb1-/- retina. Crb2, Crb3 and Crb1 interacting proteins remain localized to the sub-apical region and therefore are not sufficient to maintain cell adhesion during light exposure in Crb1-/- retinas. Thus we propose that during light exposure Crb1 is essential to maintain, but not assemble, adherens junctions between photoreceptors and Müller glia cells and prevents retinal disorganization and dystrophy. Hence, light may be an influential factor in the development of the corresponding human diseases.

TMEM16B, A Novel Protein with Calcium-Dependent Chloride Channel Activity, Associates with a Presynaptic Protein Complex in Photoreceptor Terminals

Photoreceptor ribbon synapses release glutamate in response to graded changes in membrane potential evoked by vast, logarithmically scalable light intensities. Neurotransmitter release is modulated by intracellular calcium levels. Large Ca2+-dependent chloride currents are important regulators of synaptic transmission from photoreceptors to second-order neurons; the molecular basis underlying these currents is unclear. We cloned human and mouse TMEM16B, a member of the TMEM16 family of transmembrane proteins, and show that it is abundantly present in the photoreceptor synaptic terminals in mouse retina. TMEM16B colocalizes with adaptor proteins PSD95, VELI3, and MPP4 at the ribbon synapses and contains a consensus PDZ class I binding motif capable of interacting with PDZ domains of PSD95. Furthermore, TMEM16B is lost from photoreceptor membranes of MPP4-deficient mice. This suggests that TMEM16B is a novel component of a presynaptic protein complex recruited to specialized plasma membrane domains of photoreceptors. TMEM16B confers Ca2+-dependent chloride currents when overexpressed in mammalian cells as measured by halide sensitive fluorescent protein assays and whole-cell patch-clamp recordings. The compartmentalized localization and the electrophysiological properties suggest TMEM16B to be a strong candidate for the long sought-after Ca2+-dependent chloride channel in the photoreceptor synapse.

In vivo confocal imaging of the retina in animal models using scanning laser ophthalmoscopy

Scanning-laser ophthalmoscopy is a technique for confocal imaging of the eye in vivo. The use of lasers of different wavelengths allows to obtain information about specific tissues and layers due to their reflection and transmission characteristics. In addition, fluorescent dyes excitable in the blue and infrared range offer a unique access to the vascular structures associated with each layer. In animal models, a further enhancement in specificity can be obtained by GFP expression under control of tissue-specific promotors. Important fields of application are studies in retinal degenerations and the follow-up of therapeutic intervention.

The multidrug resistance protein 1 (Mrp1), but not Mrp5, mediates export of glutathione and glutathione disulfide from brain astrocytes.

Astrocytes play an important role in the glutathione (GSH) metabolism of the brain. To test for an involvement of multidrug resistance protein (Mrp) 1 and 5 in the release of GSH and glutathione disulfide (GSSG) from astrocytes, we used astrocyte cultures from wild‐type, Mrp1‐deficient [Mrp1(–/–)] and Mrp5‐deficient [Mrp5(–/–)] mice. During incubation of wild‐type or Mrp5(–/–) astrocytes, GSH accumulated in the medium at a rate of about 3 nmol/(h.mg), whereas the export of GSH from Mrp1(–/–) astrocytes was only one‐third of that. In addition, Mrp1(–/–) astrocytes had a 50% higher specific GSH content than wild‐type or Mrp5(–/–) cells. The presence of 50 μm of the Mrp inhibitor MK571 inhibited the rate of GSH release from wild‐type and Mrp5(–/–) astrocytes by 60%, but stimulated at the low concentration of 1 μm GSH release by 40%. In contrast, both concentrations of MK571 did not affect GSH export from Mrp1(–/–) astrocytes. Moreover, in contrast to wild‐type and Mrp5(–/–) cells, GSSG export during H2O2 stress was not observed for Mrp1(–/–) astrocytes. These data demonstrate that in astrocytes Mrp1 mediates 60% of the GSH export, that Mrp1 is exclusively responsible for GSSG export and that Mrp5 does not contribute to these transport processes.